Methods for amplifying nucleic acids provide useful tools for the detection of human pathogens, detection of human genetic polymorphisms, detection of RNA and DNA sequences, for molecular cloning, sequencing of nucleic acids, and the like. In particular, the polymerase chain reaction (PCR) has become an important tool in the cloning of DNA sequences, forensics, paternity testing, pathogen identification, disease diagnosis, and other useful methods where the amplification of a nucleic acid sequence is desired. (See e.g., PCR Technology: Principles and Applications for DNA Amplification (See, Erlich, ed., 1992); PCR Protocols: A Guide to Methods and Applications (See, Innis et al., eds, 1990)).
PCR permits the copying, and resultant amplification, of a target nucleic acid. Briefly, a target nucleic acid, e.g. DNA, is combined with a sense and antisense primers, dNTPs, DNA polymerase and other reaction components. (See, Innis et al., supra) The sense primer can anneal to the antisense strand of a DNA sequence of interest. The antisense primer can anneal to the sense strand of the DNA sequence, downstream of the location where the sense primer anneals to the DNA target. In the first round of amplification, the DNA polymerase extends the antisense and sense primers that are annealed to the target nucleic acid. The first strands are synthesized as long strands of indiscriminate length. In the second round of amplification, the antisense and sense primers anneal to the parent target nucleic acid and to the complementary sequences on the long strands. The DNA polymerase then extends the annealed primers to form strands of discrete length that are complementary to each other. The subsequent rounds serve to predominantly amplify the DNA molecules of the discrete length.
In general, PCR and other methods of amplification use primers which anneal to either end of the DNA of interest. While such methods are easy when there is only a single target, and the ends are well defined and invariable, they become more difficult when there are multiple targets in which the sequences are not identical. This is a particular problem with antibody variable genes and the hypervariable regions within variable genes, especially in the creation of ligand libraries where unbiased amplification and cloning of numerous different ligands is required. Degenerate primers which anneal to regions of similarity (but not identity) at the ends of the variable gene and primers which anneal to constant sequences flanking the antibody variable gene have been used to overcome this problem. Each of these approaches, however, has disadvantages. For example, using degenerate primers may introduce foreign sequences into the priming site and also may not amplify all members of the library. Furthermore, amino acid modification as a result of these changes have adversely affected solubility, expression levels, and affinity. (See, e.g. de Haard H J, et al. (1998) Protein Eng. 11(12):1267. Honegger and Pluckthun (2001) J Mol Biol. 309(3):687.). Using primers which anneal to constant sequences flanking the antibody variable gene also typically may lead to the addition of extra conserved sequences to the variable sequences of interest, which may not be required.
The complementarity determining regions (CDRs) of immunoglobulin molecules have evolved over millions of years to specifically bind antigens within the context of the immunoglobulin molecule structure. The immunoglobulin molecule structure provides a scaffold for the CDRs so that they can extend out and contact antigen. During the maturation of antibody producing B cells, recombinations of variable region encoding genes, V, D, and J. Recombinations of V, D, and J which do not create functional antibody genes have their expression levels downregulated. As a result, most of the mRNA which can be isolated from circulating lymphocytes corresponds to functional V genes. The CDRs found within such functional V genes, and hence mRNA, thus provide a very rich source of potential binding elements. Although such CDRs could be amplified using primers annealing to the adjacent antibody framework regions, the PCR products arising from such an amplification, would always have the framework regions recognized by such primers attached to the PCR product. In applications in which CDRs would be transplanted from antibodies to another scaffold, such framework regions may interfere with the correct folding of the scaffold. Because of the high degree of variablility of the antibody hypervariable regions, there has been no method available to isolate variable or hypervariable regions, free of flanking sequences.
Thus, there is a need in the art for a method of amplifying target sequences without including regions flanking the target sequence in the amplified product or imposing primer sequences on the amplified product.